Our goal is to investigate the mechanisms involved in regulation of zinc and copper metabolism by the liver. This is approached using primary, monolayer cultures of rat liver parenchymal cells. Research has demonstrated that glucocorticoids increase the intracellular accumulation and redistribution of zinc and enhanced expression of the metallothionein (MT) gene. Glucagon augments this process. Copper accumulation is stimulated by epinephrine. Glucocorticoids increase ceruloplasmin (Cp) synthesis and secretion while epinephrine increases the efflux of cellular copper as ceruloplasmin. We are now in a position to look at the mechanisms in more detail and place them within a functional context. In the next phase, copper and zinc uptake kinetics will be further evaluated via total cellular retention, retention of the fast accumulated/rapidly exchanged cellular pool and efflux exchange. These parameters will be compared in cells from fetal rats, adrenalectomized young adult rats, old adult rats and streptozotocin-induced diabetic rats. The influences of dexamethasone, epinephrine, glucagon and vasopressin and combinations of these as well as LEM and interleukin-1 on cellular copper and zinc uptake and ceruloplasmin synthesis-secretion and MT synthesis-degradation will be examined. Inhibitions of protein synthesis with actinomycin D and cycloheximide and protein secretion with tunicamycin and leupeptin will be used to evaluate the mechanisms relating to MT and Cp. Liver cell toxins, initially bromobenzene and t-butyl hydroperoxide and Fe-induced lipid peroxidation will be evaluated for an effect on uptake kinetics, hormonal responsiveness and cellular metallothionein content and synthesis, ceruloplasmin synthesis and secretion, intracellular glutathione (GSH and GSSG), lipid peroxidation, GSH peroxidase and GSH reductase, H202 production, glucose production and 3H-proline incorporation into collagen. Changes in MT mRNA and Cp mRNA levels will be measured by 32P-cDNA hybridization and northern blotting. Cells will be made copper deficient or zinc deficient by altering the Cu and Zn content of the media. After criteria to evaluate deficiency have been satisfied, the response of deficient cells to hormonal stimulation and the ability of defient cells to handle the cell toxins will be tested. The protective effect of Cu and Zn repletion, Se, Alpha-tocophenol, 13-cis-retinoic acid and specific hormones e.g. dexamethasone will be evaluated.